EXTRACTION, ISOLATION, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITIES OF BRIDELIA FERRUGINEA BARK EXTRACTS
ABSTRACT
Medicinal plants are those plants that are used in treating and preventing specificailments and diseases that affect human beings. Hence the important role of medicinal plants in health care deliverycannot be over emphasized. Brideliaferruginea is atropical medicinal plant which belongs to the family of euphorbiaceae commonly used in traditional Africa medicine for treating various diseases. My research in this study was concentrated on the bark of bridelia ferruginea to detect the active ingredient responsible for the antimicrobial activities extraction. Phytochemicalscreening, thin layer chromatography, column chromatography, GC–MS, AAS and biological activities was carried out on the crude methanol extract of Bridelia ferruginea. The pytochemical screening for the methanol crude extract indicate the presence of tannins, alkaloids, saponins, steroids, resins and phlobatahin, while the phytochemical screening for acetone crude extract indicate the presence of tannins, alkaloids, flavonoids, phlobatanin and reducing sugar. And the photochemical tests on the hexane crude extract gave negative result all through. Isolation was done using Column chromatography, AAS was used for the determination of some certain heavy metals present in both the isolate and the crude which may be toxic to man health and the GC–MS was used to determine the presences of unknown substances in the sample. Result for GC-MS shown the presence of seven compounds which are medicinal namely, 2-[4-methyl-6-(2,6,6-trimethylcyclohex-1-enyl)hexa-1,3,5-trienyl]cyclohex-1-en-1-carboxaldehyde, limonene-6-ol,pivalate, 1-Heptatriacotanol, Benzene,1-4-dicloro, cyclobarbital, 2-nonadecanone 2,4-dinitrophenylhydrazine, 1-monolinoleylglycerol trimethylsilylether. While for AAS, two metals were detected namely iron (2.1ppm) and zinc (0.05ppm) which are non-toxic for human consumption. And also result from the antibacterial activities shows that Bridelia ferruginea was highly susceptible to Escherichia coli and highly resistant to Proteus vulgaris.
TABLE OF CONTENT
Title page……………………………………………………………………..i
Certification………………………………………………………………….ii
Dedication …………………………………………………………………..iii
Acknowledgment ……………………………………………………………iv
Abstract………………………………………………………………………v
CHAPTER ONE
1.0 Introduction……………………………………………………………..1
1.1 Micro-organisms………………………………………………………....1
1.2 Classification of micro-organism………………………………………...2
1.2.1 Bacteria………………………………………………………………...3
1.2.2 Virus…………………………………………………………………...4
1.2.3 Algae…………………………………………………………………..4
1.2.4 Fungi…………………………………………………………………..5
1.2.5 Protozoa ………………………………………………………………6
CHAPTER TWO
2.0 Literature review………………………………………………………..7
2.1 Bacteria…………………………………………………………………7
2.2 Bacteria and its cells……………………………………………………9
2.3 Shape of the bacteria…………………………………………………...12
2.4 Environmental effect of bacteria……………………………………….14
2.5 Medicinal plants………………………………………………………...16
2.5.1 Characteristics of medicinal plants…………………………………...17
2.6 Medicinal plants as antimicrobial……………………………………...26
2.7 Why considering medicinal plants as antimicrobial…………………….28
2.8 Botanical profile of Bridelia ferruginea ………………………………30
2.8.1 Taxonomy of plant…………………………………………………...31
2.8.2 Plant description…………………………………………………….31
2.8.3 Properties……………………………………………………………32
2.8.4 Cultivation details…………………………………………………...32
2.8.5 Edible uses…………………………………………………………..32
2.8.6 Medicinal uses……………………………………………………....32
2.8.7 Propagation………………………………………………………….33
2.9 Bridelia ferruginea as antimicrobial…………………………………….33
CHAPTER THREE
3.0 Materials and methods………………………………………………….35
3.1 Aims and objectives…………………………………………………….35
3.2 Materials…………………………………………………………………35
3.2.1 Chemicals,solvents and chemical reagents……………………………35
3.2.2 Equipment and materials………………………………………………36
3.3 Methods………………………………………………………………. 36
3.3.1 Collection and preparation of plant materials…………………………36
3.3.2 Extraction of plant materials…………………………………………..36
3.3.3 Phytochemical screening of Methanol extract………………………...37
3.3.4 Phytochemical screening of Hexane extract………………………….40
3.3.5 Phytochemical screening of Acetone extract…………………………43
3.4 Thin-layer chromatography……………………………………………47
3.5 Isolation and characterization of the crude extract……………………49
3.5.1 Column chromatography………………………………………………49
3.5.2 Mass of fractions gotten from the column chromatography…………..49
3.6 Instrumentation techniques………………………………………………50
3.6.1 Sample analysis for GC-MS…………………………………………..50
3.6.2 Sample analysis for AAS………………………………………………51
3.7 Anti-microbial activity of Methanol extract of Bridelia ferruginea……………51
3.7.1 Method……………………………………………………………………….51
3.8 Flow diagram of the entire experiment carried out in this
research work…………………………………………………………………..52
CHAPTER FOUR
4.0 Result and discussion ……………………………………………………………53
4.1 Result of phytochemical analysis of methanol, N-Hexane, and
Acetone extract of Bidelia ferruginea…………………………………………...53
4.2 Instrumentation techniques……………………………………………………..55
4.2.1 Result for GC-MS analysis……………………………………………………55
4.2.2 Result for AAS analysis ………………………………………………………61
4.3 Anti-microbial activities result…………………………………………………62
CHAPTER FIVE
Conclusion………………………………………………………………………….64
Recommendation…………………………………………………………………..64
Reference
CHAPTER ONE
1.0 INTRODUCTION
1.1 MICRO-ORGANISM
A microorganism or microbe is an organism (form of life) that is microscopic (too small to see by the unaided human eye) [Delong et al 2001]. Is a microscopic living organism, which may be single-celled or multicellular. The study of microorganisms is called microbiology, a subject that began with the discovery of microorganisms in 1674 by Antonie van Leeuwenhoek, using a microscope of his own design [Madigan M et al 2006].
Microorganisms are very diverse and include all bacteria, archaea and most protozoa. This group also contains some species of fungi, algae, and certain microscopic animals, such as rotifers. Many Macroscopic animals and plants have microscopic juvenile stages. Some microbiologists also classify viruses as microorganisms, but others consider these as nonliving. In July 2016, scientists reported identifying a set of 355 genes from the last universal common ancestor of all life, including microorganisms, living on Earth [Madigan M et al 2006].
Microorganisms are often described as single-celled, or unicellular, organisms; however, some unicellular protists are visible to human eye, and some multicellular species are microscopic [Delong et al 2001].
Single-celled microorganisms were the first forms of life to develop on Earth, approximately 3–4 billion years ago. Further evolution was slow, and for about 3 billion years in the Precambrian eon, all organisms were microscopic. So, for most of the history of life on Earth, the only forms of life were microorganisms. Bacteria, algae and fungi have been identified in amber that is 220 million years old, which shows that the morphology of microorganisms has changed little since the Triassic period. [Madigan M et al 2006].
Merging the idea of the microscopic and the very small with the older idea of an organism as a living entity or cell, the concept of a microorganism enabled a real appreciation of the microbial world as one that is amenable to study using similar tools and approaches even though representing distinctly different types of reproductive units and cell organizations [John A 2014].
1.2 CLASSIFICATION OF MICRO – ORGANISM
Microorganisms can be found almost anywhere in the taxonomic organization of life on the planet. Bacteria and archaea are almost always microscopic, while a number of eukaryotes are also microscopic, including most protists, some fungi, as well as some animals and plants. Viruses are generally regarded as not living and therefore not considered as microorganisms, although the field of microbiology also encompasses the study of viruses [Madigan M et al 2006].
1.2.1 BACTERIA
Bacteria constitute a large domain of prokaryotic microorganisms. Typically a few micrometers in length, bacteria have a number of shapes, ranging from spheres to rods and spirals, bacteria were among the first life forms to appear on earth, and are present in most of its habitats. Bacteria inhabit soil, water, acidic hot springs, radioactive waste, and the deep portions of earth’s crust [Rappe M.S 2003].
Most bacteria have not been characterized, and only about half of the bacterial phyla have species that can be grown in the laboratory. The study of bacteria is known as bacteriology, a branch of microbiology. There are approximately ten times as many bacterial cells in the human flora as there are human cells in the body, with the largest number of the human flora being in the gut flora, and a large number on the skin. The vast majority of the bacteria in the body are rendered harmless by the protective effects of the immune system, and some are beneficial. However, several species of bacteria are pathogenic and cause infectious diseases, including cholera, syphilis, anthrax, leprosy, and bubonic plague. The most common fatal bacterial diseases are respiratory infections, with tuberculosis alone killing about 2 million people per year, mostly in sub-Saharan Africa [WHO 2002].
In developed countries, antibiotics are used to treat bacterial infections and are also used in farming, making antibiotic resistance a growing problem. In industry, bacteria are important in sewage treatment and the breakdown of oil spills, the production of cheese and yogurt through fermentation, and the recovery of gold, palladium, copper and other metals in the mining sector, as well as in biotechnology, and the manufacture of antibiotics and other chemicals [Ishige T et al 2005].
1.2.2 VIRUS
A virus is a small infectious agent that replicates only inside the living cells of other organisms. Viruses can infect all types of life forms, from animals and plants to microorganisms, including bacteria and archaea. Viruses spread in many ways; viruses in plants are often transmitted from plant to plant by insects that feed on plant sap, such as aphids; viruses in animals can be carried by blood-sucking insects. These disease-bearing organisms are known as vectors. Influenza viruses are spread by coughing and sneezing. Viruses display a wide diversity of shapes and sizes, called morphologies. In general, viruses are much smaller than bacteria.
Most viruses that have been studied have a diameter between 20 and 300nanometres. Some filoviruses have a total length of up to 1400 nm; their diameters are only about 80nm. Most viruses cannot be seen with an optical microscope so scanning and transmission electron microscopes are used to visualise virions [Breitbart M et al 2005].
1.2.3 ALGAE
Algae is an informal term for a large, diverse group of photosynthetic organisms which are not necessarily closely related, and isthus polyphyletic. Included organisms range from unicellular genera, such as Chlorella and the diatoms, to
multicellular forms, such as the giant kelp, a large brown alga which may grow up to 50 m in length. Most areaquatic and autotrophic and lack many of the distinct cell and tissue types, such as stomata, xylem, and phloem, which are found in land plants. The largest and most complex marine algae are called seaweeds, while the most complex freshwater forms are the Charophyta, a division of green algae which includes, for example, Spirogyra and the stonewort’s. Most algae contain chloroplasts that are similar in structure to cyanobacteria. Chloroplasts contain circular DNA like that in cyanobacteria and presumably represent reduced endosymbiotic cyanobacteria [Allaby M. 1992].
1.2.4 FUNGI
Fungi is any member of a group of eukaryotic organisms that includes organisms that includes microorganisms such as yeasts and molds, as well as the more familiar mushrooms. These organisms are classified as kingdom fungi, which is separate from the other eukaryotic life kingdoms of plants and animals.
A characteristic that places fungi in a different kingdom from plants, bacteria, and some protists is chitin in their cell wall. Similar to animals, fungi are heterotrophs; they acquire their food by absorbing dissolved molecules, typically by secreting digestive enzymes into their environment [Wikipedia 2016].
The fungi have several unicellular species, such as baker's yeast and fission yeast. Some fungi, such as the pathogenic yeast, can undergo phenotypic switching and grow as single cells in some environments, and filamentous hyphae in others [Madigan M et al 2006].
Along with bacteria, fungi are responsible for the decomposition and reprocessing of vast amounts of complex organic matter; some of this is recycled to the atmosphere as CO2, while much is rendered into a form that can be utilised by other organisms [john wiley et al 2005].
1.2.5 PROTOZOA
Protozoa, as traditionally defined, are mainly microscopic organisms, ranging in size from 10 to 52 micrometers. Some, however, are significantly larger. Among the largest are the deep-sea–dwelling xenophyophores, single-celled foraminifera whose shells can reach 20 cm in diameter. Free-living forms are restricted to moist environments, such as soils, mosses and aquatic habitats, although many form resting cysts which enable them to survive drying. Many protozoan species are symbionts, some are parasites, and some are predators of bacteria, algae and other protists protozoawere defined as single-celled animals or organisms with animal-like behaviors, such as motility and predation. The group was regarded as the zoological counterpart to the "protophyta", which were considered to be plant-like, asthey are capable of photosynthesis. The terms protozoa and protozoans are now mostly used informally to designate single-celled, non-photosynthetic protists, such as the ciliates, amoebae and flagellates. The term Protozoa was introduced in 1818 for a taxonomic class, but in later classification schemes the group was elevated to higher ranks, including phylum, subkingdom and kingdom [Protozoa 2006].
CHAPTER TWO
2.0 LITERATURE REVIEW
2.1 BACTERIA
The word bacteria is the plural of the New Latin bacterium, which is the latinisation of the Greek bakterion, the diminutive of bakteria, meaning "staff, cane", because the first ones to be discovered were rod-shaped [Harper, Douglas]. The ancestors of modern bacteria were unicellular microorganisms that were the first forms of life to appear on Earth, about 4 billion years ago. For about 3 billion years, most organisms were microscopic, and bacteria and archaea were the dominant forms of life. In 2008, fossils of microorganisms were discovered and named as the Francevillian biota. Although bacterial fossils exist, such as stromatolites, their lack of distinctive morphology prevents them from being used to examine the history of bacterial evolution, or to date the time of origin of a particular bacterial species. Bacteria were also involved in the second great evolutionary divergence, that of the archaea and eukaryotes. Here, eukaryotes resulted from the entering of ancient bacteria into endosymbiotic associations with the ancestors of eukaryotic cells, which were them selves possibly related to the Archaea [Dyall SD et al 2004].
Bacteria display a wide diversity of shapes and sizes, called morphologies. Bacterial cells are about one-tenth the size of eukaryotic cells and are typically 0.5–5.0 micrometers in length. However, among the smallest bacteria are members of the genus Mycoplasma, which measure only 0.3 micrometers, as small as the largest viruses. Some bacteria may be even smaller, but these ultra microbacteria are not well-studied. Most bacterial species are either spherical, called cocci (sing. coccus, from Greek kókkos, grain, seed), or rod-shaped, called bacilli (sing. bacillus, from Latin baculus, stick). Some bacteria, called vibrio, are shaped like slightly curved rods or comma shaped; others can be spiral-shaped, called spirilla, or tightly coiled, called spirochaetes. A small number of species even have tetrahedral or cuboidal shapes. More recently, some bacteria were discovered deep under Earth's crust that grows as branching filamentous types with a star-shaped cross-section. The large surface area to volume ratio of this morphology may give these bacteria an advantage in nutrient-poor environments.
This wide variety of shapes is determined by the bacterial cell wall and cytoskeleton, and is important becauseit can influence the ability of bacteria to acquire nutrients, attach to surfaces, swim through liquids and escape predators [Wanger G et al 2008].
The bacterial cell is surrounded by a cell membrane (also known as a lipid, cytoplasmic or plasma membrane). This membrane encloses the contents of the cell and acts as a barrier to hold nutrients, proteins and other essential components of the cytoplasm within the cell. As they are prokaryotes, bacteria do not usually have membrane-bound organelles in their cytoplasm, and thus contain few large intracellular structures. They lack a true nucleus, mitochondria, chloroplasts and the other organelles present in eukaryotic cells [Berg JM et al 2002].
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